Establishment of a semicontinuous bioreactor culture of Physcomitrella patens for mass production of protoplasts

For large scale protoplast transformation of the moss Physcomitrella patens (Hedw.) B.S.G. semicontinuous protonema suspension cultures in bioreactors were established and optimised with regard to the yield of protoplasts. Supplementation of Knop medium with 2.5 mM ammonium tartrate markedly improved the protoplast yield (7.4 x 10(4) protoplasts/mg dry weight). Bioreactor culture was performed in 5 1 vessels. By harvesting on average I 100 ml/day which corresponds to a dilution rate of 0.22/d a semicontinuous culture was maintained for 49 days yielding 51 1 of suspension culture. Beginning eleven days after the start of the bioreactor run around 6 % of the cells were polyploid (4C) which might indicate aging of the culture caused by phytohormone accumulation.